Sub-acute Toxicological Evaluation of Methanol Whole Plant ...
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Journal of Pharmaceutical Development and Industrial Pharmacy Volume 1 (3), 2019
Available online at www.jpdip.com
ISSN: 2659 - 1472
Research Article
Sub-acute Toxicological Evaluation of Methanol Whole
Plant Extract of Tapinanthus dodoneifolius (D.C) Danser in
Wistar Rats
V. Mamman 1, B. Mahmud1, M.G. Magaji1, N.M. Danjuma1 1Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Ahmadu Bello University
Zaria, Kaduna State
Corresponding Author:
B. Mahmud
Email: [email protected]
Received: 16 November 2019
Revised and Accepted: 15 December 2019
Abstract Tapinanthus dodoneifolius (Loranthaceae), has diverse ethnomedicinal uses which include the management of mental illness, epilepsy, diarrhoea,
malaria, pain and hypertension. Despite these uses and many validated pharmacological properties, the safety of T. dodoneifolius is yet to be
evaluated. This study therefore, aimed to evaluate the sub-acute toxicity profile of methanol whole plant extract of T. dodoneifolius in rats.
Phytochemical screening of the plant extract was carried out according to standard protocols. Oral median lethal dose (LD50) was estimated using
the Organisation for Economic Cooperation and Development (OECD) 423 limit test. Doses of 250, 500 and 1000 mg/kg of the extract were
administered for 28 consecutive days using the OECD 407 guideline. The body weights of animals were taken at 7 days interval within 28 days
period. Thereafter, animals were sacrificed and blood samples collected for haematological, biochemical and electrolyte analysis. The spleen, heart,
liver, kidney, lungs and stomach were also collected for histological examinations. The extract contains alkaloids, cardiac glycosides, saponins,
tannins, flavonoids, steroids, terpenoids and carbohydrate. The LD50 was found to be ≥ 5000 mg/kg. The extract showed significant (p≤0.05)
increase in body weight and decrease in alkaline phosphatase (ALP). There was no significant difference in the mean organ weight. The histological
studies did not reveal any serious abnormalities in the organs examined. The methanol whole plant extract of T. dodoneifolius does not possess the
toxic effects that could affect its ethnomedicinal uses.
Keywords: Tapinanthus dodoneifolius; toxicity; body weight; organ weight; haematological indices; biochemical parameters
1. INTRODUCTION
The increasing use of medicinal plants in the treatment
of many ailments necessitates the toxicological
screening to ascertain their safety among users [1].
Toxicity is a term that describes the level of an adverse
effect caused by the interaction of a toxicant and
cell(s) of a living organism [2]. Medicinal plants are
essential part of healthcare delivery system in resource
poor areas of the world. About 80% percent of the
world’s population uses herbal medicine in their
healthcare delivery system [3]. The greater demands
for resources and time in evaluating the safety and
standardizing the use of medicinal plants have led to
much emphasis of screening them for efficacy based
on traditional claims rather than safety. Toxicological
evaluation of medicinal plants is as important as
pharmacological validation of ethnomedicinal claims
[4]. Other reason that may have led to the shift of
attention more on pharmacological validation may
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stem from the general belief of safety and acceptability
of medicinal plants.
Tapinanthus dodoneifolius is a bushy parasitic plant
that grows on a wide range of trees around the
savannah zone. The plant belongs to the family
Loranthaceae. T. dodoneifolius is an important plant
use in the treatment of many diseases such as stomach
ache, diarrhoea, dysentery, diabetes, epilepsy,
hepatitis, hypertension, wounds, cancer etc among the
Hausa and Fulani tribes of northern Nigeria. The local
names of the plants among different tribes in Nigeria
are Etu-lonchi (Nupe), Elozie (Ibo), Kauchi (Hausa)
and Afomu Igba (Yoruba) [5]. Other ethnomedicinal
uses of Tapinanthus dodoneifolius are treatment of
inflammation, fever, infection, dizziness, energy loss,
irritability, vertigo and headache. The has a broad
spectrum of activity against multidrug resistant
bacteria and fungal isolates of farm animals [6, 5]. T.
dodoneifolius is also reported to have larvicidal and
molluscidal effects. Related Africa mistotle
Agelanthus dodonefolins has been reported to have
antiplasmodial activity [7]. Despite the dependence on
medicinal plants most especially in resource poor
areas, there is still sketchy information about their
safety [8]. Tapinanthus dodoneifolius is one of such
plant. This study was undertaken to evaluate sub-acute
toxicity profile of methanol whole extract of
Tapinanthus dodoneifolius in Wistar rats.
2. MATERIALS AND METHODS
Collection of Plant
The whole plant of Tapinanthus dodoneifolius was
collected from the bush in Sabon Gari Local
Government Area of Kaduna State, Nigeria in the
month of June, 2018. It was identified and
authenticated by Mr. Namadi Sanusi of the
Department of Botany, Ahmadu Bello University,
Zaria. A voucher number; 0370 was obtained by
comparing with existing specimen.
Preparation of Plant Extract
The whole plant of Tapinanthus dodoneifolius was
washed, air-dried under shade after which it was
grounded to coarse powder using pestle and mortar.
About 900 g of the powdered plant material was
extracted with 7 L of 70 %v/v methanol for three days
using cold maceration with intermittent shaking. The
mixture was then filtered using Whatman filter No 1.
The filtrate was concentrated and subjected to air
drying in crucible. Percentage yield of the methanol
extract was calculated and the obtained extract was
stored in a desiccator and subsequently referred to as
Methanol Extract of Tapinanthus dodoneifolius
(METD). Fresh concentrations of the extract were
prepared on each day of the experiment by
reconstituting in distilled water.
Experimental Animals
Wistar rats of both sexes were obtained from the
Animal House Facility of the Department of
Pharmacology and Therapeutics Ahmadu Bello
University, Zaria. Animals were housed in cages with
rodent diet and water ad libitum. Ethical approval with
an approval number: (ABUCAUC/2019/006) was
obtained from the Ahmadu Bello University
Committee on Animal Use and Care (ABUCAUC).
Phytochemical Screening
Preliminary phytochemical screening of the methanol
whole extract of Tapinanthus dodoneifolius was
carried out according to methods described by Trease
and Evans and Ayoola [9, 10]
Acute Toxicity Study
The acute toxicity study was carried out according to
the Organization for Economic Co-operation and
Development (OECD) guidelines 423[11] and fixed
dose studies was adopted with 5000 mg/kg body
weight as the limit dose. Three rats were fasted for 3-
4 hours, and each rat was administered 5000 mg/kg of
methanol whole plant extract of Tapinanthus
dodoneifolius. Food was withheld for 1 hour post drug
administration. Each rat was observed individually for
the first 30 minutes and periodically during the first 4
hours and then daily for 14 days for any sign of
toxicity, such as tremor, convulsion, salivation,
lacrimation, diarrhea, lethargy, sleep, respiratory,
behavior pattern, onset and recovery from toxicity and
death.
Sub-acute Oral Toxicity Study
The method described by the Organization for
Economic Co-operation and Development (OECD)
test guideline 407 was adopted for the study [12].
Twenty-four rats (12 males and 12 females) were
weighed and grouped into 4 of 3 male and 3 female
rats in each group (the males were separated from the
females). Group 1, 2, 3 and 4 received 10 mL/kg distil
water, 250, 500 and 1000 mg/kg per oral of the extract
respectively for 28 consecutive days. Animals were
sacrificed under chloroform anaesthesia after being
deprived of pellet but had free access to water for 24
hours. Blood samples were collected through cardiac
puncture into EDTA and non-heparinized containers
for haematological and biochemical analysis
respectively. The liver, heart, spleen, stomach, kidney
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and lungs were collected, weighed and stored in 20 mL
of 10 % formyl saline sample bottles (Axiom,
Zhanjiang Gong Jong medical technology Co. Ltd,
China) and thereafter processed for histopathological
studies.
Body Weight
The body weights of the animals were taking at weekly
interval during the course of the 28-days treatment.
The weight changes were calculated in respect to the
initial body weights on day zero.
Organ Weight Index
The heart, lungs, stomach, liver, spleen and kidney
were collected, freed of connective tissues and rinsed
in normal saline, blotted with filter paper and weighed.
The relative organ body weight (ROW) was calculated
as: ROW= (organ weight/total body weight) × 100.
Haematological Analysis
Blood samples were collected and assayed. The
parameters assayed include haematocrit (HCT),
Hemoglobin (HB), red blood cell (RBC) count,
platelet count (PLT), white blood cell (WBC) count,
monocytes (MON), lymphocytes (LYMPH),
granulocytes (GRAN) using automated haematology
analyzer (Mythic 18 by Orphee, Switzerland).
Biochemical Analysis
Serum blood samples were analyzed for alkaline
phosphatase (ALP), alanine transaminase (ALT),
aspartate transaminase (AST), albumin (Alb), total
protein (TP), urea (Bu), creatinine (Crea) using (
Gesan Chem 200, USA) automated machine.
Histopathology
The kidney, liver, heart, lungs, stomach, and spleen
were placed into 10 % formalin fixative for
histological examinations. Tissue slide were viewed at
a magnification of x250 and photomicrographs of the
tissues were obtained with the aid of a consultant
histopathologist
Statistical Analysis:
Data were expressed as mean ± SEM. One-way and
spilt plot analysis of variance (ANOVA) were
performed appropriately to compare the differences
between means followed by Bonferoni post hoc tests
using SPSS version 20.0. A mean difference was
considered significant when p ≤ 0.05.
3. RESULTS
The percentage yield of the powdered plant was
14.4%.
Phytochemical Constituents
The preliminary phytochemical screening of METD
revealed the presence of alkaloids, cardiac glycosides,
saponins, tannins, flavonoids, steroids/triterpenes,
terpenoids and carbohydrates. However,
anthraquinones was absent.
Acute Toxicity Study
The methanol whole plant extract produced no adverse
effect in rats at the dose of 5000 mg/kg. There were no
changes in behavior, nature of stool and saliva. There
was no mortality observed in the different groups of
rats after 7 days. The oral median lethal dose (LD50) of
the plant was estimated to be ≥ 5000 mg/kg.
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Figure 1: Effect of 28 days Administration of Methanol Extract of Tapinanthus dodoneifolius on Body Weight
of Wistar Rats
Values are expressed as Mean ± S.E.M; *p≤0.05 compared to control; Split plot ANOVA followed by Bonferroni post
hoc test, n=6, METD = Methanol Extract of Tapinanthus dodoneifolius
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Table 1: Effect of 28 days Administration of Methanol Extract of Tapinanthus dodoneifolius on Some
Haematological Parameters in Wistar Rats
Treatment Groups
Parameters Control 250 mg/kg 500 mg/kg 1000 mg/kg
WBC (×103/µL) 6.72±0.91 6.10±0.77 5.27±0.89 5.74±0.71
Hb (g/dL) 13.11±1.11 13.33±0.46 13.03±0.69 14.49±0.59
RBC (×106/µL) 4.85±0.08 4.65±0.14 4.65±0.14 4.65±0.14
PLT (×103/µL) 306.40±31.09 400.67±25.72 323.47±34.93 205.80±21.20
HCT (%) 40.72±3.75 41.52±1.05 39.98±2.09 43.34±1.66
MON (%) 11.00±0.95 9.13±1.24 7.18±1.67 5.16±0.72*
LYMPH (%) 33.54±2.14 36.92±2.51 32.47±1.93 32.36±2.59
GRAN (%) 55.62±2.41 53.15±2.36 44.85±2.83* 44.64±1.52*
Values are expressed as Mean ± S.E.M., *p≤0.05 compared to control - One way ANOVA followed by Bonferroni
post hoc test, n=6, METD: Methanol extract of Tapinanthus dodoneifolius, WBC: White blood Cell, RBC: Red blood
cells, HGB: Haemoglobin, HCT: Haematocrit, PLT: Platelets, LYMP: Lymphocytes, MON: Monocytes, GRAN:
Granulocytes
Table 2: Effect of 28 days Administration of Methanol Extract of Tapinanthus dodoneifolius on Serum
Biochemical Parameters in Wistar Rats
Treatment Groups
Parameters Control 250 mg/kg 500 mg/kg 1000 mg/kg
ALT (IU/L) 13.00±3.08 9.00±1.53 9.50±1.53 15.60±1.36
AST (IU/L) 143.20±18.83 95.00±13.95 156.17±8.45 148.20±6.42
ALP (IU/L) 35.24±4.77 27.62±3.86 26.03±2.52 20.48±1.96*
TP (g/dL) 9.22±0.68 10.04±0.78 12.33±1.01 12.05±0.88
ALB (g/dL) 3.16±0.18 2.26±0.24 2.90±0.09 3.02±0.23
Values are expressed as Mean ± S.E.M., *p≤0.05 compared to control; One way ANOVA followed by Bonferroni post
hoc test, n=6, ALT: Alanine amino transferase, AST: Aspartate amino transferase, ALP:Alkaline Phosphatase,
TP:Total protein, ALB: Albumin, METD: Methanol extract of Tapinanthus dodoneifolius, IU: International unit
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Table 3: Effect of 28 days Administration of Methanol Extract of Tapinanthus dodoneifolius on Kidney
Function in Wistar Rats
Treatment Group
Parameters Control 250 mg/kg 500 mg/kg 1000 mg/kg
Urea (mg/dL) 46.08±1.28 48.25±2.67 44.05±1.62 49.96±0.95
Sodium (mmol/L) 114.54±5.17 121.95±4.47 123.55±3.08 106.32±5.12
Potassium (mmol/L) 16.82±2.42 11.82±0.76 17.00±1.06 13.96±1.22
Creatinine (mEq/L) 0.94±0.07 0.92±0.06 0.88±0.07 0.82±0.05
Chloride (mEq/L) 21.80±1.39 22.40±1.54 25.33±1.91 25.33±1.9
Bicarbonate (mg/dL) 88.20±3.77 84.00±7.31 85.83±.2.40 85.40±5.95
Values are expressed as Mean ± S.E.M; One way ANOVA followed by Bonferroni post hoc test, n=6, METD:
Methanol extract of Tapinanthus dodoneifolius
Table 4: Effect of 28 days Administration of Methanol Extract of Tapinanthus dodoneifolius on Mean Organ
Weight in Wistar rats
Treatment Groups
Organs Control 250 mg/kg 500 mg/kg 1000 mg/kg
Liver 3.78±0.14 3.22±0.12 3.41±0.13 3.73±0.14
Kidney 0.74±0.05 0.56±0.05 0.63±0.04 0.66±0.05
Lungs 0.97±0.19 0.71±0.11 0.69±0.06 0.69±0.06
Heart 0.42±0.04 0.36±0.06 0.36±0.02 0.41±0.02
Spleen 0.55±0.10 0.39±0.02 0.45±0.03 0.49±0.06
Stomach 1.28±0.12 1.10±0.05 1.21±0.10 1.18±0.07
Values are expressed as Mean ± S.E.M; One way ANOVA followed by Bonferroni post hoc test, n=6, METD =
Methanol extract of Tapinanthus dodoneifolius
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Figure 2: Photomicrographs of Heart Sections (H & E stained at ×250 magnification) of Rats Following 28
days oral administrations of Methanol Extract of Tapinanthus dodoneifolius
A (Control); B (250 mg/kg); C (500 mg/kg); D (1000 mg/kg) Sections showing normal cardiac muscles (M)
Figure 3: Photomicrographs of Kidney Sections (H & E stained at ×250 magnification) of Rats Following 28
days Oral Administrations of Methanol Extract of Tapinanthus dodoneifolius
A (Control); B (250 mg/kg); C (500 mg/kg); D (1000 mg/kg) T (Normal kidney tubules and glomerulus); TN (Slight
tubular necrosis); LH (Lymphocyte hyperplasia)
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Figure 4: Photomicrographs of Liver Sections (H & E stained at ×250 magnification) of Rats Following 28
days Oral Administrations of Methanol Extract of Tapinanthus dodoneifolius
A (Control); B (250 mg/kg); C (500 mg/kg); D (1000 mg/kg) H (Normal hepatocytes); HN (moderate hepatocyte
necrosis)
Figure 5: Photomicrographs of Lung Sections (H & E stained at ×250 magnification) of Rats Following 28
days Oral Administrations of Methanol Extract of Tapinanthus dodoneifolius
A (Control); B (250 mg/kg); C (500 mg/kg); D (1000 mg/kg) A (normal lung alveoli); AC (slight alveoli
congestion); HP (nuclei hardening and pyknosis)
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Figure 6: Photomicrographs of Spleen Sections (H & E stained at ×250 magnification) of Rats Following 28
days Oral Administrations of Methanol Extract of Tapinanthus dodoneifolius
A (Control); B (250 mg/kg); C (500 mg/kg); D (1000 mg/kg) N (normal red and white pulp); LH (Lymphocyte
hyperplasia)
Figure 7: Photomicrographs of Stomach Sections (H & E stained at ×250 magnification) of Rats Following 28
days Oral Administrations of Methanol Extract of Tapinanthus dodoneifolius
A (Control); B (250 mg/kg); C (500 mg/kg); D (1000 mg/kg) M (normal mucosa epithelium of the stomach); LH
(lymphocyte hyperplasia)
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4. DISCUSSION
The use of medicinal plants like Tapinanthus
dodoneifolius in the treatment of human diseases has
enjoyed global acceptability [13]. Comprehensive
knowledge about the safety of the plant is yet to be
examined despite numerous pharmacological
beneficial activities. Therefore, the present study was
carried out to ascertain the acute and sub-acute toxicity
of Tapinanthus dodoneifolius in experimental animals.
The methanol whole plant extract of Tapinanthus
dodoneifolius was found to be non-toxic since there
was no sign of toxicity and mortality at 5000 mg/kg.
The phytochemical constituents such as alkaloids,
cardiac glycosides, saponins, tannins, flavonoids,
steroids/triterpenes, terpenoids and carbohydrates
present in the extract may be responsible for the
observed pharmacological activity [14]. A non-
significant increase in body weight (Figure 1).was
observed which may be due to the nutritional benefit
of the plant extract [15]
Haematological parameters are used to ascertain the
level of safety of compounds or extract when exposed
to human [16, 17] and has been used to explain
haematologically-related functions [18]. Changes in
haematological parameters are necessary to risk
examination due to their predictive values of toxicity
[19]. The non-significant changes in RBC, HCT, Hb,
and PLT level by METD (Table 1) show that the plant
does not affect erythropoiesis and osmotic fragility of
the red blood cells [20]. The decrease in the level of
monocytes and granulocytes could suggest that the
extract may exert toxic effect on the immune system
and cause agranolocytosis at dose of 1000 mg/kg and
above. The white blood cells (WBC) are the first line
of defence which responds to infectious agents,
inflammatory process or tissue injury.
The methanol whole plant extract of Tapinanthus
dodoneifolius did not show significant changes in AST
and ALT enzymes (Table 2), ALP was decreased
significantly at dose of 1000 mg/kg suggesting
inhibition of the enzyme by the extract.
The serum biochemical assay is used to assess renal
and hepatic functions in order to find possible
pathological changes due to the extract. Renal and
hepatic function analysis is highly useful in the
toxicity screening of compounds including medicinal
plants as both are important for the excretion and
metabolism of drugs by an organism [21]. The liver
contains host of enzymes (ALT, AST, ALP) that serve
as biochemical markers of liver damages. Once there
is a liver damage, these enzymes leak into the serum
and show increased activities [22].
The extract at all doses tested could not significantly
alter the level of total protein (Table 2). This implies
that it is non-toxic to the kidneys and liver. Serum
protein and albumin are sensitive indicators of liver
function. They are synthesized and metabolized in the
liver and serve as a means of assessing hepatocellular
injury [23]. Total protein measurement reflects
nutritional status and may be used to screen and help
diagnose kidney, liver disease and many other
conditions. Low total protein levels suggest a liver,
kidney disorder or any other condition in which
protein is not digested or absorbed properly. High total
protein level may be seen with chronic inflammation
or liver infections [24].
Measurements of urea, creatinine and uric acid are
used to assess renal dysfunction and their normal
values indicate absence of renal damage [25]. From the
result obtained (Table 3), there was no significant
difference among the extract treated groups compared
to the control. This suggests that the extract does not
seem to possess toxic effect on the kidney. Decrease
in organ weight is an expression of toxicity resulting
from exposure to toxic substances [26]. The liver;
kidney, lungs, stomach, spleen and heart were not
adversely affected compared to the control group,
throughout the 28 day treatment period (Table 4).
Hence, the whole plant extract is nontoxic to the
organs in the sub-acute study.
There were no any adverse abnormalities in their gross
examinations of the heart, kidney, stomach, spleen,
lungs and liver isolated in various group. The
histological studies on these organs by the extract did
not reveal any pathological changes after treatment
compared to the control when administered for 28
days as seen in plate figures 2, 3, 4, 5, 6 and 7.
5. CONCLUSION
The methanol whole plant extract of T. dodoneifolius
does not seem to possess the toxic effects that could
affect its ethnomedicinal use.
ACKNOWLEDGEMENT
The Authors are grateful to the entire staff of the
Department of Pharmacology and Therapeutics,
Ahmadu Bello University, Zaria, Nigeria for
providing the enabling environment to carry out this
work.
CONFLICT OF INTERESTS
There are no conflicts of interest.
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REFERENCES
1. Asgarpanah J, Ramezanloo F. Chemistry,
pharmacology and medicinal properties of
Peganum harmala L. African Journal of.
Pharmaceutical Pharmacology 2012;
6(22):1573-1580
2. Syahmi ARM, Vijayarathna S, Sasidharan S,
Latha, LY, Kwan YP, Lau YL, Shin LN,
Chen Y. Acute oral toxicity and brine shrimp
lethality of Elaeis guineensisjacq. (Oil palm
leaf) methanol extract. Molecules2010;
15:8111– 8121.
3. Peter JH. The Role of Plants in Traditional
Medicine and Current Therapy. The Journal
of Alternative and Complementary Medicine.
1995; 1(2): 131-143.
4. Sofowora A. Medicinal plants and
Traditional Medicine in Africa.2nd edition.
Spectrum Books Ltd, Ibadan, Nigeria; 1993.
289 p.
5. Deeni YY, Sadiq NM. Anti-microbial
Properties and Phytochemical constituents of
Tapinanthus dodoneifolius (DC) Danser
(Loranthaceae): An Ethno medicinal Plant of
Hausa land, Northern Nigeria. Journal of
Ethnopharmacology 2002; 83(3):235-40.
6. Cepleanu F, Hamburger MO, Sordat B.
Screening of tropical medicinal plants for
molluscidal, larvicidal, fungicidal and
cytotoxic activities and brine shrimp toxicity.
International Journal of Pharmacognosy
1994; 32:294-307.
7. Builders MI, Oguru MO, Aguiyi C. Anti-
plasmodial potential of the African mistletoe:
Agelanthus dodoneifolius Polh and Wiens.
Indian Journal on Pharmaceutical Sciences
2012; 74(3):223-229.
8. World Health Organization (WHO).
Traditional Medicine Fact sheet 2008; No.
134. Retrieved from:
www.who.int/mediacentre/factsheets/fs134/
en/
9. Evans, W.C. (2002). Trease and Evans
Pharmacognosy. 15th Edition, Elsevier,
India. p. 27, 46, 183-184, 289-291, 411-413,
434, 485-486.
10. Ayoola GA, Coker HAB, Adesegun SA,
Adepoju-Bello AA, Obaweya K, Ezennia
EC, Atangbayila TO. Phytochemical
Screening and Antioxidant Activities of some
Selected Medicinal Plants used for Malarial
Therapy in South Western Nigeria. Tropical
Journal of Pharmaceutical Research 2008;
7:1019-1024.
11. Organisation for Economic Cooperation and
Development (OECD). Guidelines for the
Testing of Chemicals/Section 4: Health
Effects Test No. 423. Acute oral toxicity-
acute toxic class method, Paris. 2002.
12. Organisation for Economic Cooperation and
Development (OECD). OECD guidelines for
testing of chemicals: Guideline 407:
Repeated dose 28-day oral toxicity in
rodents. Office of Economic and
Community Development, Paris. 2008.
13. Afolabi SO, Akindele AJ, Awodele O,
Anunobi CC, Adeyemi OO. A 90 day chronic
toxicity study of Nigerian herbal preparation
DAS-77 in rats. Complementary and
Alternative Medicine 2012; 12:79.
14. Edeoga HO, Okwu E, Mbaebie BO.
Phytochemical constituents of some Nigerian
medicinal plants. African Journal of
Biotechnology2005; 4(7):685-688.
15. Ezeonwumelu JOC, Julius AK, Muhoho CN,
Ajayi AM, Oyewale AA, Tanayen JK,
Balogun SO, Ibrahim A, Adzu B, Adiukwu
CP, et al. Biochemical and Histological
Studies of Aqueous Extract of Bidens pilosa
leaves from Ugandan Rift Valley in
Rats.British Journal of Pharmacology and
Toxicology2011; 2(6):302-309.
16. Agbaje EO, Adeneye AA, Daramola AO.
Biochemical and Toxicological Studies of
Aqueous Extract of Syzigium Aromaticum
(L.) Merr. & Perry (Myrtaceae) in Rodents.
African Journal
ofTraditional,Complementary Alternative
Medicine2009; 6(3): 241–254.
17. Ibrahim MB, Sowemimo AA, Sofidiya MO,
Badmos KB, Fageyinbo MS, Abdulkareem
FB, Odukoya OA.Sub-acute and chronic
toxicity profiles of Markhamia tomentosa
ethanolic leaf extract in rats. Journal of
Ethnopharmacology 2016; 193:68-75.
18. Yakubu MT, Akanji MA and Oladiji AT.
Haematological evaluation in male albino
rats following chronic administration of
aqueous extract of Fadogia agrestis stem.
Pharmacognosy Magazine 2007; 3: 34– 38.
19. Olso H, Betton G, Robinson D,Thomas K,
Monro A, Kolaja G, Lilly P, Sanders J Sipes
G, Bracken W, Dorato M, et al. Concordance
of the toxicity of pharmaceuticals in humans
and in animals. Regulation of Toxicology and
Pharmacology2000; 32, 56–67.
Mamman et al. J Pharmaceutical Development and Industrial Pharmacy. 1(3), 2019, 16-27
27
Available online at www.jpdip.com
20. Guyton AC, Hall JE.
TextbookofMedicalPhysiology,
11thed.Elsevier Saunders, USA. 2006.1152 p
21. Olorunnisola OS, Bradley G and Afolayan
AJ. Acute and subchronic toxicity studies of
methanolic extract of Tulbaghia violacea
rhizomes in Wistar rats.African Journal of
Biotechnology2012; 11: 14934–14940.
22. Akanji MA, Nafiu MO, Yakubu MT.
Enzyme activities and histopathology of
selected tissues in rats treated with potassium
bromate. African Journal on Biomedical
Research 2008; 11: 87-95.
23. Ganong WF. Review of Medical Physiology.
20th edition. New York: Lange Medical
Books, McGraw Hill Companies
International. 2016. p. 500–515
24. Ighodaro I, Silvanus IS, Vincent O, Lucy A.
Chronic Toxicity Studies of Aqueous Leaf
Extract of Voacanga africana in Wistar Rats.
Journal of Appl. Sci. Environ. Management
2015; 19 (4) 639-646.
25. Davis ME and Bredt ND. Renal methods for
toxicity, in Principles and Methods of
Toxicology, Raven Press. New York, USA.
1994
26. Adeyemi OO, Akindele AJ, Nwumeh KI.
Acuteandsubchronictox- icological
assessmentof Byrsocarpus coccineus Schum.
And Thonn. (Con-naraceae) aqueous leaf
extract. International Journal of Applied
Research Natural Product 2010; 3, 1–11.